Glycodendrimers of polypropyletherimine

ABSTRACT

A glycodendrimer comprising: a) a non-toxic dendrimer polypropyletherimine core supporting 16 terminal carboxylic acid groups, and b) conjugated to said core 2, 3, 4 or 5 glucosamine molecules, wherein each glucosamine is linked directly through a zero length amide bond with a residue of a terminal carboxylic acid group. The invention also extends to defined populations comprising said glycodendrimer molecules, pharmaceutical compositions comprising said molecules or populations, process for preparing the molecules and formulations, and therapeutic uses of the molecules, populations and compositions.

The present application claims priority from U.S. provisionalapplication 61/344,571 filed 24 Aug. 2010 and GB application number1109292.1 filed 2 Jun. 2011 both of which are incorporated herein byreference.

The present disclosure relates to glycodendrimers containing apolypropyletherimine core, pharmaceutical formulations comprising same,use of each thereof in treatment, particularly in the treatment ofconditions mediated by pro-inflammatory cytokines, such as Interleukin-6and Interleukin-8 The disclosure further relates to processes for theproduction of the glycodendrimers, and use of polypropyletherimine coresfor preparation of said glycodendrimers.

BACKGROUND

In 2009, Park et al defined the structural basis of the recognition ofLPS by TLR4-MD2 [1,2].

In brief, MD2 has a hydrophobic pocket that is lined by a charged,hydrophilic entrance. Lipid A (which consists of two phosphorylatedglucosamine molecules linked together via an ester bond) binds to theentrance of this pocket. Its lipid chains then enter MD2's hydrophobicpocket. The TLR4-MD2-LPS complex undergoes a conformational change andTLR4 dimerizes. Intracellular signalling follows.

The importance of the saccharide portions of LPS and of electrostaticinteractions between LPS and MD2 have recently been reinforced [3,4].

The most relevant residue is thought to be Tyr102, followed by Lys91,Arg96, Arg106, Asn114 and Ser118. These residues and/or residues intheir very close proximity had already been identified as having animportant role in the recognition of LPS by MD2 [1,3,4].

Bacterial infections and surgical tissue injury trigger the same cellsurface receptor-ligand interactions that are based upon TLR4immuno-modulation. This does not involve a single receptor-ligandinteraction. Rather, these pro-inflammatory cytokine responses aremediated by polyvalent receptor-ligand interactions between bacteriallyderived lipopolysaccharide (LPS) and/or surgically derived hyaluronanfragments and the cell surface TLR4 receptor [5]. The binding affinityof these ligands for this receptor increases exponentially as the numberof receptor-ligand interactions increases. Therefore, it is desirable toadapt the concept of polyvalency to novel biomaterial design by creatingnew biomaterials that can interact with and modulate tissue injurypathways.

As polyvalency requires multiple and co-operative receptor-ligandinteractions, pharmacological intervention will also require newmedicines that are based upon molecules that are also capable ofmultiple and co-operative interactions. This has already been achievedwith protein-based medicines, which interact with multiple cell surfacereceptors with high affinity. For many years the aim has been to achieveanalogous co-operative interactions with synthetic macromolecules.However, it has been found that in biological systems, the use of linearpolymers has been much less successful than anticipated. Attempts to uselinear polymers have been impeded by:—(1) the structural heterogeneityof the macromolecules used; (2) an inability to control their size andmolecular weight characteristics; and (3) the toxic side effects ofactivating complement and coagulation triggered pathways.

In addition, in the case of linear polymers displaying saccharides, theyhave a tendency to self-associate and to form micelles because of theamphiphilic characteristics of many polymer-saccharide combinations. Inthe case of polysaccharides, their structural heterogeneity and thecomplex nature of the chemistry involved in their preparation hasimpeded the manufacturing scalability and reproducible synthesis ofdefined oligosaccharide-like molecules with the appropriate biologicalproperties. In general, many synthetic steps are required, and the polarnature of the chemical intermediates and products make them difficult topurify. These compounds are also difficult to handle because they tendto be hygroscopic syrups, chemically labile, susceptible to rapidmicrobial degradation, and difficult to process into medicines. Thesefundamental problems have impaired the scale up manufacture ofsaccharide based macromolecules for pharmaceutical use.

WO 03/089010 disclosed certain glycodendrimers based on the PAMAM core.Generation three PAMAM dendrimers conjugated to glucosamine orglucosamine sulphate to form a 3.5 generation dendrimer have beenextensively studied. These molecules have been shown to have veryinteresting biological activity and low toxicity, and in particular havesignificant anti-cytokine and anti-chemokine properties.

However, these compounds have not been progressed as pharmaceuticalproducts and have never been dosed to a human because, to date, no wayhas been identified to commercially and viably manufacture them to alevel of “purity” that is suitable for administration to a human. Whatis more many of the so called impurities are very closely relatedchemical species to the desired species and thus separation of thedifferent entities is very difficult and may not be possible usingcurrently available chromatography and/or filtration based techniques.

Whilst not wishing to be bound by theory it is now believed by thepresent inventors that the chemistry on which PAMAM glycodendrimers isbased is inherently incompatible with providing a molecule suitable forpharmaceutical use.

Nevertheless PAMAM glycodendrimers are special molecules because oftheir biological activity. Whilst many cores exist from which dendrimerscan be made it seems that glycodendrimers made from alternative cores donot possess the requisite biological properties.

The present inventors believe that, surprisingly, the glycodendrimersdescribed herein are likely to provide biological properties to renderthem suitable for pharmacological intervention and additionally that thechemistry on which the molecules according to the invention are based issuitable for providing a molecule which can ultimately be used as apharmaceutical product.

SUMMARY OF THE INVENTION

Thus there is provided a glycodendrimer comprising:

a) a non-toxic dendrimer polypropyletherimine core supporting 16terminal carboxylic acid groups, and

b) conjungated to said core 2, 3, 4 or 5 glucosamine molecules,

wherein each glucosamine is linked directly through a zero length amidebond with a residue of a terminal carboxylic acid group.

DETAILED DESCRIPTION OF THE INVENTION

Dendrimers are a class of polymeric compounds that can be distinguishedfrom conventional linear polymers by their highly branched, circular andsymmetrical architecture.

The first dendrimers were made by divergent synthesis approaches byVogtle in 1978, Denkewalter at Allied Corporation in 1981, DonaldTomalia at Dow Chemical in 1983 and in 1985 and by Newkome in 1985. In1990, a convergent synthetic approach was introduced by Jean Fréchet.Dendrimer popularity then greatly increased, resulting in more than5,000 scientific papers and patents by 2005 [6].

Divergent synthesis is essentially where the molecules are built up inlayers from the centre outwards. The reference to generations is inessence a reference to the number of layering steps in the synthesis toproduce the dendrimer. Thus in the divergent approach, the dendrimer isgrown outwards from the core, typically there is a doubling of thenumber of reactive functionalities with each new “generation”. However,care needs to be taken with the terminology, generation, becausedifferent starting materials require different techniques of synthesis,which can mean that a dendrimer of a certain “generation” made from onestarting material may, in some ways, not necessarily be directlycomparable to a dendrimer prepared from a different starting material,even though nominally they are given the same numerical generation. Theterm generation does not equate to absolute physical dimensions of thedendrimer.

In contrast, convergent synthesis is where the molecules are built infragments and assembled as the last step or at a late stage ofsynthesis. Thus, the convergent growth method involves the synthesis ofdendritic wedges possessing carbohydrates as one of the structuralcomponents, followed by the linking of these wedges to furthercomponents that provide branching, and then, finally, the attachment ofthese dendrons to the core component to obtain the desired dendrimer.The adoption of this convergent synthesis protocol typically results inlarger quantities of the saccharide being displayed on the surface ofthe dendrimer in particular it may result in a complete saccharidecapping on the dendrimer when the final dendrimer is assembled.Dendrimers prepared convergently are not assigned a generation.

Advantageously, dendrimers have a molecular structure that can be muchmore precisely defined than is possible for linear polymers [6,7].

As mentioned above, different starting materials can be used to generatethe core. Dendrimers based on polyamidoamine (PAMAM) have beenextensively studied. PAMAM dendrimers are prepared by divergentsynthesis. Detailed reviews of divergently synthesised anioniccarboxylic acid terminated PAMAM dendrimers can be found in WO 03/089010and [8].

PAMAM dendrimers are formed by the incremental addition of branchedlayers called generations onto a core. Typically, they are available inwhole generations which are amine terminated, and half-generations whichare carboxylic acid terminated. The generation of a dendrimer istherefore representative of both its size (measured as its diameter inangstroms) and its molecular weight [9] in relative terms.

Divergent dendrimers can also be synthesised from polypropyleneimine(PPI), polylysine, triazine and polypropyletherimine A detailed reviewin 2010 of the current field of divergently synthesised dendrimers andtheir applications can be found in [10]. There has been some evidence tosuggest that dendrimers which terminate in free carboxylic acids, asopposed to those terminating in amine groups, have improvedtoxicological profiles [11].

Dendrimers have been applied to a number of fields. In thepharmaceutical field they have been investigated as drug carriers and indiagnostic applications. Very rarely have the dendrimers been consideredtherapeutic entities in their own right, partly because of their sizeand complex nature.

In the application of dendrimers as a drug carrier or as a diagnostictool the field seems to have been obsessed with producing larger andlarger dendrimers. There are many synthetic chemistry papers on thematter. However, it should be noted that it has been repeatedlydemonstrated in the literature that high valency (in these largemolecules) does not correspond with high biological affinity. In fact,the precise nature of the underlying scaffold is as important as thenumber of copies of saccharide ligand per molecule for molecules thatare biologically active [13]. That is to say the core is not simply aninert support for the amino sugar molecule.

WO 03/089010 discloses a generation 3.5 PAMAM dendrimer (64 terminalcarboxylic acids) with a partially glycosylated surface which hasimmuno-modulatory properties. About 15% or less of the surface terminalcarboxylic acids are linked to glucosamine by a zero length amide bond.In 2004, Shaunak showed that this dedndrimer blocked pro-inflammatorycytokine responses [8,14] including inhibited the release of thepro-inflammatory cytokines TNF-alpha, IL-1 beta and IL-6 from primaryhuman monocytes, macrophages and dendritic cells by highly purified LPS.

Whilst this PAMAM glycodendrimer molecule has some interesting in vitroand in vivo biological activity, including the prevention of excessivescarring in animal models, this molecule has not been progressed as atherapeutic agent (FIG. 1) because of the inability to make the moleculein a suitable form for use as a pharmaceutical.

Our analysis of the difference between the biologically activeglycodendrimers and the biological inactive glycodendrimers leads us tobelieve that the interaction with specific amino acids residues at theentrance to the cavity of MD2 is important for the biological activity.

We also believe that the hydrophilic entrance of the pocket on MD2 (towhich Lipid A binds) is blocked by the glycodendrimer according to thepresent disclosure and in particular the dendrimer glucosamine Thisprevents TLR4 dimerization and signalling events.

Our studies have shown that the biologically active partiallyglycosylated dendrimer shows the largest number and the strongestinteractions with several of the residues lining the entrance to MD2'spocket. Several of these residues are also important for the binding ofLPS to MD2. The residues with the highest normalized interaction valueswere Lys91, Tyr102, Arg106, Asn114 and Ser118. Several other residues,with lower interaction values, also contributed significantly to theco-operative binding of the partially glycosylated dendrimer to humanMD2; they were Arg96, Ser98, Lys109, Thr112 and Thr116. Thus in oneembodiment the dendrimers of the present disclosure interact with one ormore (for example 1, 2, 3, 4 or 5) residues selected from Lys91, Tyr102,Arg106, Asn114 and Ser118.

Interact as employed herein refers to non-covalent bonding, for examplehydrogen bonds, ionic bonds, van der Waals forces and/or hydrophobicinteractions.

In one embodiment the dendrimer of the present disclosure interacts withLys91. In one embodiment the dendrimer of the present disclosureinteracts with Tyr 102. In one embodiment the dendrimer of the presentdisclosure interacts with Arg106. In one embodiment the dendrimer of thepresent disclosure interacts with Asn114. In one embodiment thedendrimer of the present disclosure interacts with Ser118. In oneembodiment the dendrimer of the present disclosure interacts with: Lys91and Tyr102; Lys91 and Arg106; Lys91 and Asn114; Lys91 and Ser118; Tyr102and Arg106; Tyr102 and Asn114; Tyr102 and Ser118; Arg106 and Asn114;Arg106 and Ser118, or Asn114 and Ser118.

In one or more embodiments the dendrimer of the disclosure also has atleast one interaction (for example 2, 3, 4 or 5) with a residue selectedfrom Arg96, Ser98, Lys109, Thr112 and Thr116.

In addition, the affinity of a partially glycosylated dendrimer forhuman MD2 was demonstrated by the increased number of close contacts(i.e., 1.3 Å) between these two molecules that involved both theglucosamine molecules and several of the dendrimer's peripheralcarboxylic acid branches. These electrostatic interactions occluded theentrance to human MD2's hydrophobic pocket and are thought to blockedaccess of the lipid chains of LPS.

It is hypothesised that the biological activity of the dendrimer surfaceresults from the presence of both the amino sugar (or sulfate thereof)and the free peripheral carboxylic acids that are from the coredendrimer.

Advantageously partially glycosylated dendrimers, according to thepresent disclosure are both flexible and dynamic, and have a hydrophilicperiphery, this means that conformational changes could induce shapecomplementarity. Thus the interaction of the glycodendrimer according tothe present disclosure with the biological target, may induce“conformational changes” in the molecule. This is thought to enable thedendrimer's surface saccharides such as glucosamine molecules to blockthe entrance of human MD2's pocket.

Additional co-operative electrostatic interactions with some of thedendrimer's free carboxylic acid branches follow. Collectively, theseinteractions as thought to block the entry of the lipid chains of LPSinto human MD2's pocket, and also prevent TLR4-MD2-LPS cell surfacecomplex formation. The biologically important outcome is that thepro-inflammatory cytokine cascade is not initiated.

Thus in one embodiment the dendrimer according to the present disclosurehas a close interaction with one or more of said amino acid residues inthe target, for example those listed above. Close as employed herein isintended to refer to an interaction of 2 Å or less such as 1.5 Å orless, in particular 1.3 Å.

Taken together, the results of our modelling studies and our biologicalstudies suggest that the interaction of the G3.5 partially glycosylatedPAMAM dendrimer (and more than likely the glycodendrimers according tothe invention) with human MD2 is specific, and that a dendriticarchitecture is important for this molecule's biological activity[15,16,17,18, 18a]. Our results also suggest that at least twoglucosamine molecules, linked via the arms of a dendrimer, are requiredto bind to several of the exposed and charged amino acids that line theentrance of the cavity on MD2. Thus the glycodendrimers of the presentdisclosure will generally comprise at least 2 glucosamine molecules(glucosamines).

It is thought that electrostatic interactions over a 20 Angstrom rangeare responsible for this interaction between dendrimer for exampledendrimer glucosamine and MD2 rather than hydrogen bond interactions(which can only occur over a distance of 4 Angstroms).

It is hypothesised that the optimum separation of the two sugar moietiessuch as the two glucosamine residues for this high affinity binding isprobably 10 Å. The binding of dendrimer, for example dendrimerglucosamine to the hydrophilic entrance of the pocket on MD2 almostcompletely occludes the entrance to the pocket in MD2; it also inducesconformational changes in MD2 itself that make it very difficult for LPSto bind to the protein as an effective agonist. Advantageously thisreduces pro-inflammatory cytokine production. This important newmechanistic observation shows that dendrimer aminosaccharides—withoutany lipid chains or phosphate groups attached to the dendrimer—can actas partial antagonists of the binding of LPS to the charged, hydrophilicentrance of the hydrophobic pocket on MD2.

Whilst not wishing to be bound by theory it is thought that the surfaceproperties (outer arm flexibility and charge distribution) and size ofthe molecule are vital to the biological activity, in particular that acombination of the amino sugar and free carboxylic acids and for examplethe cluster density such as the zero length amide bond formed arerequired on a suitably sized scaffold to block the target receptor andgenerate the therapeutic results. The hydrophilic surface of theglycodendrimer also has an important role to play.

Compounds able to produce co-operative effects are very important inmodulating biological control mechanisms in the immune system. What ismore they are seldom realised in purely synthetic molecules. Thedendrimers of the present disclosure are suitable for producingco-operative effects in vitro and in vivo

Whilst the data generated from the generation 3.5 anionic carboxylicacid terminated PAMAM dendrimer molecule has been very promising andexciting, one reason for its lack of progression is that despiterepeated attempts by many workers in the field, to date, it has not beenpossible to prepare the molecule as a “monodispersion”, in essence acharacterisable population of molecules that can be reproduciblyprepared to the satisfaction of the drug regulators. To date, no methodis available for generating a PAMAM dendrimer as a charactarisablepopulation that can reproducibly prepared to the satisfaction of drugregulators. One may say that the chemistry is inherently flawed in thatit is not possible to prevent side reactions occurring during thesynthesis of this molecule. The intrinsic problems are caused byincomplete reactions, and cyclization reactions with PAMAM chemistry,which means that there will always be a significant degree of structuralheterogeneity to the final PAMAM product.

Thus the inventors have searched for a replacement molecule which sharesthe characteristics of the generation 3.5 PAMAM dendrimer molecule.However, it turns out that the surface characteristics, size,flexibility and hydrophilicity that provides the desirable biologicalprofiles are not so easy to replicate.

The inventors have now identified a very limited number ofglycodendrimers with the desirable biological properties, and with acore based on different chemistry which, with care, can be used to makea molecule that has the purity and the reproducibility of scale upmanufacture and will meet the degree of analytical chemicalcharacterisation required of a pharmaceutical drug, for example togenerate a monodispersion that is substantially a single chemicalentity.

The core is based on polypropyletherimine and surprisingly theglycodendrimers made employing this core seem to at least share theadvantageous biological properties of the PAMAM glycodendrimer and may,in one or more areas, show improvements over the PAMAM glycodendrimer.

The term “monodispersion” is used as per the meaning generallyunderstood in the field of dendrimer chemistry. This means that thedendrimer has a narrow molecular weight distribution in which oneparticular species of a defined molecular weight is predominantlypresent. More specifically, the one particular species is present at 90%or more, for example 91, 92, 93, 94, 95, 96, 97, 98% or more.

In one embodiment the glycodendrimer of the present invention is amixture of a small number of well defined chemical entities which areinnocuous or substantially similar to the main dendrimer species and forexample have similar biological activity.

Thus in one embodiment the molecules of the disclosure will be providedas a population of molecules. The population may comprise a number ofdiscrete molecules some of which may contain more or less than twoglucosamines Generally the number of molecules with less than twoglucosamines is low, for example less than 3%, such as 2%, 1% or less,in particular 0.5% or 0.1% w/w. In one embodiment the total number ofmolecules with more than 4 glucosamines is low for example less than 3%,such as 2%, 1% or less, in particular 0.5% or 0.1% w/w. In oneembodiment the number of molecules with 3 or 4 glucosamines is 50% w/wor less, for example 40%, 30%, 20%, 10% or 5% w/w or less.

Innocuous in the context of the present specification is intended torefer to an impurity that causes no deleterious effects and isessentially harmless in the biological context. Deleterious effects may,for example, relate to tissue toxicity or catalysis or degradation orany other property that could be considered a disadvantage for amedicine.

Substantially similar as employed herein is intended to refer to wherethe molecule comprises the same components as the desired species butthey are present in a different ratio, for example substantially similarwill generally refer to a glycodendrimer comprising the same core andsugars but the number of carboxylic acids on the core and/or the numberor of sugars conjugated to the core is different to that which isdesired.

In one embodiment the dendrimer core is prepared divergently and thusthe resulting dendrimer can be assigned a generation. In one embodimentthe generation of the core dendrimer is 3.

Dendrimer core as employed herein is intended to refer to the brancheddendrimer polymer before the surface is modified by the conjugation ofthe glucosamine thereto. Generally, if the final core is beingconsidered then it will terminate in free carboxylic acids. However, ifthe core is at an intermediate stage it may terminate with a functionalgroup other than a carboxylic acid.

Terminal carboxylic acid group as employed herein is intended to referto a free carboxylic acid group —C(O)OH, located at the end of onesurface branch of the dendrimer and any carboxylic acid residues.

Residue of a terminal carboxylic acid as employed herein is intended torefer to a portion of the terminal carboxylic acid left after a chemicalreaction with another entity, such as the amino sugar for example—C(O)—.

Free carboxylic acid group is intended to refer to the unreacted(unconjugated carboxylic acid) —C(O)OH.

Glycodendrimer as employed herein is intended to refer to the entityresulting from conjugating the glucosamine to some of the terminalcarboxylic acids on the dendrimer core.

The glucosamine is linked to the dendrimer core by an amide bond formedby a nitrogen in the glucosamine with a carbonyl from a terminalcarboxylic acid group. This is a direct amide bond, also referred to asa zero length amide bond.

For a given population of molecules the numbers of carboxylic acidspresent may be calculated as an average over the whole population.

In one embodiment the population is defined in that it will not includemolecules with carboxylic acids below a defined lower threshold andabove a defined upper threshold, for example where 16 carboxylic acidcontained molecules are required the lower limit may be 12 and the upperlimit may be 20 or even 18.

In one embodiment a glycodendrimer comprises a corresponding number ofterminal carboxylic acids and carboxylic acid residues to the totalnumber of free carboxylic acids in the starting (before conjution). Thatis to say the combined number of terminal carboxylic acids andcarboxylic acid residues corresponds to the number of carboxylic acidsin the starting core. In the glycodendrimer molecules of the presentdisclosure there is at least one and generally more than one freecarboxylic acid, for example where the number of free carboxylic acids=X−Y, wherein X is a number in the range 12 to 20 (and corresponds tothe number of carboxylic acids of the dendrimer core) and Y is a numberin the range 1 to 5 (and corresponds the number of surface conjugatedsugar molecules).

In one embodiment the number of free carboxylic acids after conjugationin a given molecule is 11, 12, 13 or 14, for example 14. The inventionalso extends to a population of glycodendrimers comprising moleculeswith 11, 12, 13 or 14 free carboxylic acids. These free carboxylic acidsare likely to have a role to play in facilitating the enhancedpolyvalent binding of the glycodendrimer to the target receptor andhence are relevant to the biological activity of the molecules.

The glycodendrimers according to the disclosure may be provided as adiscrete population of the molecules that this characterisable.

Advantageously, carboxylic acid terminated anionic polypropyletheriminedendrimers have a remarkable lack of toxicity in vitro compared tocationic polypropyletherimine dendrimers [19]. This inherent toxicity ofhigher generation cationic dendrimers means that they are unlikely to besuitable or safe for repeated intravenous administration as apharmaceutical drug in man [11]. Thus the glycodendrimers of the presentdisclosure are believed to have low toxicity, which renders themsuitable for use as a pharmaceutical.

In one embodiment 2, 3, 4 or 5 glucosamines are conjugated to thedendrimer core, for example a core containing on average 16 carboxylicacids, for example 2, 3, or 4, such as 2 or 3. In one embodiment 2glucosamines are conjugated to the dendrimer core.

In one embodiment there is provided a population of molecules with onaverage 2, 3, 4 or 5 glucosamines are conjugated to the dendrimer core,for example a core containing on average 16 carboxylic acids, forexample 2, 3, or 4, such as 2 or 3.

In one embodiment on average 2, 3, 4, or 5 glucosamines are conjugatedto the dendrimer core. This population may comprise molecules withbetween 1 and 8 glucosamines conjugated thereto. However, the latterwill generally be in a minority, for example less than 10% w/w or 5% w/wor less, such as 3%, 2% or 1% or less.

In one embodiment the main species of glycodendrimer comprises 2glucosamines but the population may, for example also comprise entitieswith 1, 3, 4 and/or 5 glucosamines, such as 1 or 3.

Even in a glycodendrimer population of molecules with the same number ofglucosamines conjugated thereto the molecules may be present asregion-isomers. That is to say the relative positions of theglucosamines in particular glycodendrimers may vary. That is to say thespatial arrangement of the glucosamines may differ in one moleculecompared to another molecule in the population.

In one embodiment the number of region-isomers of the mainglycodendrimer species is minimised, in particular, for a givenpopulation of glycodendrimers with the same number of glucosaminesconjugated thereto at least 50% of said population will be the desiredregion-isomer, in particular 75% will be the desired region-isomer.

The region-isomer distribution may be influenced to provide the desiredoutcome by optimising the synthetic chemistry conditions duringconjugation.

In one embodiment 33% or less of the dendrimers carboxylic acids areconjugated to a glucosamine, for example 29%, 28,%, 27%, 26%, 25%, 24%,23%, 22%, 21%, 20%, 19%, 18,%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the dendrimers carboxylicacids are conjugated, such as 18.75 or 12.5%.

In one embodiment the number of conjugated carboxylic acids is in therange 3% to 14%, such as 6% to 13%, in particular 12.5%.

However the current inventors believe that at least two glucosamines arerequired in each molecule to effectively block the target receptor.

Furthermore in one aspect there is provided a highly optimizedglycodendrimer comprising 2 glucosamines and 14 remaining freecarboxylic acids. In one embodiment the dendrimer is a population ofdendrimers where the later entity is the major component, for example asa monodispersion.

This molecule (i.e., generation 3 anionic carboxylic acid terminatedpolypropyletherimine dendrimer glucosamine) is physically smaller thanthe generation 3.5 PAMAM dendrimer glucosamine previously described inthe art. The generation 3.5 PAMAM dendrimer had 64 terminal carboxylicacids in the core.

Surprisingly, this small polypropyletherimine dendrimer has only 16terminal carboxylic acids in the core. At the same concentration (on amg/ml basis), it is believed that the use of polypropyletherimine coresfor preparation of said glycodendrimers can reduce some pro-inflammatorycytokines to an equal or a greater extent than the generation G3.5 PAMAMdendrimer glucosamine as summarised in Table 1 below.

TABLE 1 Summary table of results from several of the figures shown. Itquantifies the fold reduction in pro-inflammatory cytokines with G3polypropyletherimine dendrimer glucosamine when compared to G3.5 PAMAMdendrimer glucosamine. The 100% control = stimulation of cytokinerelease from cells that were stimulated with 25 ng/ml LPS. Drugconcentration - tested in human cells (μg/ml) Cytokine 200 100 50 2512.5 TNF-α G3 polypropyletherimine nd 390 570 807 477  dendrimerglucosamine G3.5 PAMAM 112 84 7 2 nd dendrimer glucosamine IL-8 G3polypropyletherimine nd 165 192 288 17 dendrimer glucosamine G3.5 PAMAM 9 10 2 1 nd dendrimer glucosamine MIP-1β G3 polypropyletherimine nd 165193 288 17 dendrimer glucosamine G3.5 PAMAM  54 53 6 2 nd dendrimerglucosamine IL-6 G3 polypropyletherimine nd 75 40 40  2 dendrimerglucosamine G3.5 PAMAM 835 910 178 5 nd dendrimer glucosamine (nd = notdone)

This small and optimized generation 3 (G3) polypropyletheriminedendrimer glucosamine may be particularly useful from a pharmaceuticalperspective because it will be more cost effective to manufacture inrespect of the starting materials employed to manufacture the same, andthe ease of execution of the chemical steps required to manufacture thefinal medicinal product.

The dendrimer core is a polypropyletherimine core. Usually this is basedon units of 3-amino-propa-1-ol. Depending on how the dendrimer issynthesised, there can be an oxygen atom at the centre of the core or anitrogen atom at the centre of the core. For details ofpolypropyletherimine dendrimers with nitrogen at the core see [20]. Fordetails of polypropyletherimine dendrimers with oxygen at the core seethe following papers [19,21,22].

In one embodiment the core is a polypropyletherimine based on 3-amino-propan-1 -ol. These dendrimers can have an oxygen atom or anitrogen atom at the very core of the molecule.

In one embodiment the dendrimer has an oxygen atom at the very core ofthe molecule.

In one embodiment the dendrimer has a nitrogen atom at the very core ofthe molecule.

The glycodendrimers herein are referred to as a polypropyletherimineglycodendrimers.

These new anionic polypropyletherimine glycodendrimers are thought tobind to the cell surface TLR4 receptor on monocytes and macrophages anddendritic cells to reduce the production of the pro-inflammatorycytokines IL-6, TNF-alpha, IL-8 and/or IL-1 beta in response to LPS orhyaluronan fragments.

Surprisingly, these dendrimers cores, when used, provide the correctcombination of features to support a 30% or less such as a 20% or lessloading of glucosamine and to provide the biological activity. It isespecially surprising that the small generation polypropyletherimineglycodendrimers have advantageous biological activity.

In one embodiment there is provided a glycodendrimer wherein theglycodendrimer is a generational dendrimer. In one embodiment there isprovided a glycodendrimer wherein the dendrimer core is a generation 3.

In one embodiment there is provided a generation 3 anionic carboxylicacid terminated (i.e., 16 peripheral carboxylic acid groups)polypropyletherimine glycodendrimer (such as glucosamine glycodendrimer)with a 12.5% surface loading of glucosamine (i.e. 2 glucosaminemolecules) with a zero length amide bond between the dendrimer core andthe glucosamine. In one embodiment the two glucosamine molecules arepresent on opposite sides of the surface of the dendrimer as shown inFIG. 4 (that is to say spatially separated to be the maximum distanceapart).

In one embodiment there is provided a generation 3 anionic carboxylicacid terminated (i.e., 16 peripheral carboxylic acid groups)polypropyletherimine glucosamine glycodendrimer with a 18.75% surfaceloading of sugar such as glucosamine (i.e. 3 glucosamine molecules) witha zero length amide bond between the dendrimer core and the glucosamine.

Thus in one embodiment there is provided a generation 3 anioniccarboxylic acid terminated (i.e., 16 peripheral carboxylic acid groups)polypropyletherimine glycodendrimer (such as glucosamine glycodendrimer)with a 25% surface loading of glucosamine (i.e. 4 glucosamine molecules)with a zero length amide bond between the dendrimer core and the sugarsuch as glucosamine.

Thus in one embodiment there is provided a generation 3 anioniccarboxylic acid terminated (i.e., 16 peripheral carboxylic acid groups)polypropyletherimine glycodendrimer (such as glucosamine glycodendrimer)with a 31.25% surface loading of sugar such as glucosamine (i.e. 5 sugarmolecules such as glucosamine molecules) with a zero length amide bondbetween the dendrimer core and the sugar such as glucosamine

The invention also provides a population of glycodendrimers wherein theaverage properties of the population are as defined herein. In oneembodiment there is provided a composition comprising a population ofgeneration 3 anionic carboxylic acid termination polypropyletherimineglycodendrimer molecules bearing 2 or 3 glucosamines.

In one embodiment there is provided a composition comprising a mixtureof generation 3 anionic carboxylic acid termination polypropyletherimineglycodendrimer molecules bearing 3 or 4 sugars, such as 3 or 4glucosamines.

The ratio of molecules bearing different numbers of sugar molecules, forexample molecules bearing 2 glucosamines and molecules bearing 3glucosamines (or the molecules with 3 glucosamines and molecules with 4glucosamines) in the mixture is in the range 1 to 99%: 99 to 1%respectively and may for example be 50:50, 75:25 or 25:75 etc.

In one embodiment there is provided a composition comprising a mixtureof generation 3 anionic carboxylic acid termination polypropyletherimineglycodendrimer molecules bearing 4 or 5 sugars, such as 4 or 5glucosamines.

In one embodiment the glycodendrimer according to the disclosure has 13or 14 free carboxylic acids, such as 14. In one embodiment theglycodendrimer according to the disclosure has 13 free carboxylic acids.In one embodiment the glycodendrimer according to the disclosure has 12free carboxylic acids. In one embodiment the glycodendrimer according tothe disclosure has 11 free carboxylic acids.

Opposite sides of the molecule as employed herein is intended to referto diametrically opposed glucosamines, or a similarly thermodynamicallyand sterically favourable conformation. It may be that the divergentapproach to the synthesis of dendrimer glucosamine leads to thefavourable addition of 2 glucosamine molecules to two of the 16carboxylic acid groups of a generation 3 anionic carboxylic acidterminated polypropyletherimine dendrimer. Typically, we believe the twoglucosamine molecules are situated at diametrically opposite ends of thesurface of the dendrimer [15,16,17].

This is also provided a dendrimer with a ratio of combined terminalcarboxylic acids/acid resides to the number of glucosamines conjugatedthereto is in the range 8:1 to 6:1, in particular 8:1.

In one embodiment the amino sugars such as glucosamines are evenlyspaced on the surface of the dendrimer.

Evenly spaced as employed herein is intended to refer to the fact thatthe glucosamines are spread across the surface of the dendrimer in abalanced manner and are not clumped together in one or more isolatedlocations on the surface.

Advantageously, dendrimers are hyperbranched, wherein the ends of eachbranch define the molecular surface of the dendrimer. Notably, (1) theirphysico-chemical properties are similar to those of conventional smallmolecule drugs; (2) they can be modified to exist as zwitterions atphysiological pH; and (3) they have a considerable buffering capacitythat makes them physico-chemically “similar” to blood proteins (e.g.,albumin), and therefore biocompatible. However, unlike proteins, they(1) do not undergo proteolytic degradation in plasma; (2) are notimmunogenic; (3) are not toxic after repeated intravenousadministration; (4) can be optimized for their circulation time; and (5)show preferential accumulation in tissues containing inflammatory cellscompared to healthy tissue at a ratio of 50:1. In addition, the NationalCancer Institute's Nanotechnology Characterisation Laboratory recentlyundertook detailed chemical and toxicological characterization ofanionic PAMAM dendrimers and found them to be both stable andbiocompatible (see the Nanotechnology Characterisation Laboratory NCI(2006) Dendrimer-based MRI contrast agentswebsite—http.//ncl.cancer.gov/working_technical_reports.asp).

Advantageously, the glycodendrimers of the present disclosure arestable, in that they are suitable for storage under appropriateconditions before use, for example use as a therapeutic agent.

In a further aspect, the present invention provides a pharmaceuticalformulation comprising a polypropyletherimine glycodendrimer of theinvention and optionally a pharmaceutically acceptablecarrier/excipient.

In one embodiment the formulation comprises 10 μg to 1 g ofglycodendrimer of the present disclosure.

The compounds and formulations of the invention are suitable foradministration parenterally for example intravenously, subcutaneously,intramuscularly, intraperitoneally and intraocularly; orally; topicallyincluding by aerosol, for example intranasally, by pulmonaryadministration, directly to the eye, transdermally (skin) such as via animpregnated plaster or a skin patch, in particular to the surface of theskin transdermal by a slow release preparation; and intramucosally forexample by buccal or rectal administration, for example as a rectalenema wherein the compound is formulated in a suitable carrier such asan aqueous carrier.

In one embodiment the formulation is suitable for topicaladministration.

In one embodiment the formulation is suitable for infusion or directinjection.

In one embodiment the formulation is suitable for oral administration.

Topical administration as employed herein includes administration toorally to the GI tract and colon etc, wherein the compound administeredis not absorbed systemically.

In another aspect, the invention provides a pharmaceutical compositioncomprising, as active ingredient, a compound of the disclosure or apharmaceutically acceptable derivative thereof in association with apharmaceutically acceptable excipient, diluent and/or carrier for use intherapy, and in particular, in the treatment of human or animal subjectssuffering from a condition susceptible to amelioration by anantimicrobial compound.

An active ingredient as employed herein is intended to refer to apharmacologically effective ingredient, for example which aretherapeutically efficacious. Examples of active ingredients includecorticosteroids, for example fluticasone propionate, fluticasonefuroate, mometasone furoate, dexamethasone, cortisone, hydrocortisone,betamethasone, prednisolone; non-steriodal anti-inflammatories forexample aspirin, ibuprofen, naproxen.

There is further provided by the present disclosure a process ofpreparing a pharmaceutical composition, which process comprises mixing acompound of the disclosure or a pharmaceutically acceptable derivativethereof, together with a pharmaceutically acceptable excipient, diluentand/or carrier.

The compounds of the disclosure may be formulated for administration inany convenient way for use in human or veterinary medicine and thedisclosure therefore includes within its scope pharmaceuticalcompositions comprising a compound of the disclosure adapted for use inhuman or veterinary medicine. Such compositions may be presented for usein a conventional manner with the aid of one or more suitableexcipients, diluents and/or carriers. Acceptable excipients, diluentsand carriers for therapeutic use are well known in the pharmaceuticalart, and are described, for example, in Remington's PharmaceuticalSciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice ofpharmaceutical excipient, diluent and/or carrier can be selected withregard to the intended route of administration and standardpharmaceutical practice. The pharmaceutical compositions may compriseas—or in addition to—the excipient, diluent and/or carrier any suitablebinder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Preservatives, stabilisers, dyes and even flavouring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may also be used.

For some embodiments, the agents of the present disclosure may also beused in combination with a cyclodextrin. Cyclodextrins are known to forminclusion and non-inclusion complexes with drug molecules. Formation ofa drug-cyclodextrin complex may modify the solubility, dissolution rate,bioavailability and/or stability property of a drug molecule.Drug-cyclodextrin complexes are generally useful for most dosage formsand administration routes. As an alternative to direct complexation withthe drug the cyclodextrin may be used as an auxiliary additive, e. g. asa carrier, diluent or solubiliser. Alpha-, beta- and gamma-cyclodextrinsare most commonly used and suitable examples are described in WO91/11172, WO 94/02518 and WO 98/55148.

The compounds of the disclosure may be milled using known millingprocedures such as wet milling to obtain a particle size appropriate fortablet formation and for other formulation types. Finely divided(nanoparticulate) preparations of the compounds of the invention may beprepared by processes known in the art, for example see InternationalPatent Application No. WO 02/00196.

The routes for administration (delivery) include, but are not limitedto, one or more of: oral (e. g. as a dry powder/free flowing particulateformulation, tablet, capsule, or as an ingestible solution orsuspension) rectal, buccal, and sublingual. The compositions of thedisclosure include those in a form especially formulated for parenteral,oral, buccal, rectal, topical, implant, ophthalmic, nasal orgenito-urinary use. In one aspect of the invention, the agents aredelivered orally, hence, the agent is in a form that is suitable fororal delivery.

In some instances it may be possible to deliver the compounds of thedisclosure by a topical, parenteral (e. g. by an injectable form) ortransdermal route, including mucosal (e. g. as a nasal spray or aerosolfor inhalation), nasal, gastrointestinal, intraspinal, intraperitoneal,intramuscular, intravenous, intrauterine, intraocular, intradermal,intracranial, intratracheal, intravaginal, intracerebroventricular,intracerebral, subcutaneous, ophthalmic (including intravitreal orintracameral).

There may be different composition/formulation requirements depending onthe different delivery systems. By way of example, the pharmaceuticalcomposition of the present disclosure may be formulated to be deliveredusing a mini-pump or by a mucosal route, for example, as a nasal sprayor aerosol for inhalation or ingestable solution, or parenterally inwhich the composition is formulated in an injectable form, for deliveryby, for example, an intravenous, intramuscular or subcutaneous route.Alternatively, the formulation may be designed to be delivered by bothroutes. Where appropriate, the pharmaceutical compositions can beadministered by inhalation, in the form of a suppository or pessary,topically in the form of a lotion, solution, cream, ointment or dustingpowder, by use of a skin patch, orally in the form of tablets containingexcipients such as starch or lactose, or in capsules or ovules eitheralone or in admixture with excipients, or in the form of elixirs,solutions or suspensions containing flavouring or colouring agents, orthey can be injected parenterally, for example intravenously,intramuscularly or subcutaneously.

For buccal or sublingual administration the compositions may beadministered in the form of tablets or lozenges which can be formulatedin a conventional manner.

For parenteral administration, the compositions may be best used in theform of a sterile aqueous solution which may contain other substances,for example enough salts or monosaccharides to make the solutionisotonic with blood.

If a compound of the present disclosure is administered parenterally,then examples of such administration include one or more of:intravenously, intraarterially, intraperitoneally, intrathecally,intraventricularly, intraurethrally, intrasternally, intracranially,intramuscularly for example as a bolus fomulation or subcutaneouslyadministering the agent, and/or by using infusion techniques.

Formulations for parenteral administration may be provided in alyophilised form for reconstitution with a water of injection orinfusion or an isotonic solution, such as glucose.

The compounds of the disclosure can be administered (e. g. orally ortopically) in the form of tablets, capsules, ovules, elixirs, solutionsor suspensions, which may contain flavouring or colouring agents, forimmediate-, delayed-, modified-, sustained-, pulsed- orcontrolled-release applications.

The compounds of the disclosure may also be presented for human orveterinary use in a form suitable for oral or buccal administration, forexample in the form of solutions, gels, syrups, mouth washes orsuspensions, or a dry powder for constitution with water or othersuitable vehicle before use, optionally with flavouring and colouringagents.

Solid compositions such as tablets, capsules, lozenges, pastilles,pills, boluses, powder, pastes, granules, bullets or premix preparationsmay also be used. Solid and liquid compositions for oral use may beprepared according to methods well known in the art. Such compositionsmay also contain one or more pharmaceutically acceptable carriers andexcipients which may be in solid or liquid form.

The tablets may contain excipients such as microcrystalline cellulose,lactose, sodium citrate, calcium carbonate, calcium sulphate, dibasiccalcium phosphate and glycine, mannitol, pregelatinised starch, cornstarch, potato starch, disintegrants such as sodium starch glycollate,croscarmellose sodium and certain complex silicates, and granulationbinders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose(HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia.

Additionally, lubricating agents such as magnesium stearate, stearicacid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers ingelatin or HPMC (hydroxypropyl methylcellulose) capsules. Preferredexcipients in this regard include microcrystalline cellulose, lactose,calcium carbonate, calcium sulphate, dibasic calcium phosphate and,mannitol, pregelatinised starch, corn starch, potato starch or highmolecular weight polyethylene glycols. For aqueous suspensions and/orelixirs, the agent may be combined with various sweetening or flavouringagents, colouring matter or dyes, with emulsifying and/or suspendingagents and with diluents such as water, ethanol, propylene glycol andglycerin, and combinations thereof.

Capsules, may be filled with a powder (of medicament alone or as blendwith selected filler(s)) or alternatively a liquid, each comprising oneor more compounds of the invention and a carrier. Where the capsule isfilled with a powder the compounds of the invention and/or the carriermay be milled or micronised to provide material with an appropriateparticle size.

Compounds of the disclosure may be coated, for example with an entericcoating when administered orally as a tablet or capsule. The tablet orcapsule, as appropriate, may, for example be coated by a thin film suchas a EUDRAGIT® film available from Rohm Pharma Polymers, which allowscontrolled dissolution in the gastrointestinal tract. The films areavailable as cationic polymers such as EUDRAGIT® E 100 (aminoalkylmethacylate copolymers) or as anionic acrylic polymers such as EUDRAGIT®L (methacrylic acid copolymers) and EUDRAGIT S.

Permeable acrylic polymers such as EUDRAGIT® RL (amino methacrylatecopolymer) and EUDRAGIT® RS are also available.

These coating formulations may be prepared as an aqueous dispersionincluding optional ingredients such as talc, silicone antifoam emulsion,polyethylene glycol. Alternatively the coating formulation may beprepared as an organic polymer solution.

Alternatively, tablets may be coated using OPADRY® (Surelease®) coatingsystems, available from Colorcon. Aqueous systems generally comprise upto 15% w/w of OPADRY®. Organic solvent systems generally comprise up to5% w/w of OPADRY®.

The coatings may be prepared by known techniques, for example by: 1.weighing the required quantity of OPADRY® film coating system, 2.weighing the required quantity of water or other solvent(s) into amixing vessel, 3. with a mixing propeller in the centre of the vesseland as close to the bottom of the vessel as possible, stirring thesolvents to form a vortex without drawing air into the liquid, 4.steadily and quickly adding the OPADRY® powder to the vortex, avoidingpowder flotation on the liquid surface, 5. increasing the stirrer speedin order to maintain the vortex, if required, and 6. after all thepowder ingredients have been added, reducing the mixer speed andcontinuing mixing for approximately 45 minutes.

Coatings can be applied by known techniques, using tablet coatingmachines. The thickness of the coating applied is generally in the range5 to 35 microns such as 10 to 30 microns, more specifically 10 or 20microns, depending on the required effect.

Alternatively, the tablet or a capsule, as appropriate, may be filledinto another capsule (preferably a HPMC capsule such as Capsugel®) toprovide either a tablet in capsule or capsule in capsule configuration,which when administered to a patient yields controlled dissolution inthe gastrointestinal tract thereby providing a similar effect to anenteric coating.

Thus in one aspect the disclosure provides a solid dose formulation of acompound of invention for example where the formulation has an entericcoating.

In another aspect the disclosure provides a solid dose formulationcomprising a protective capsule as outer layer, for example as a tabletin a capsule or a capsule in a capsule. The enteric coating may providean improved stability profile over uncoated formulations.

The compounds of the disclosure may also be administered orally, inveterinary medicine, in the form of a liquid drench such as a solution,suspension or dispersion of the active ingredient together with apharmaceutically acceptable carrier or excipient.

The compounds of the invention may also, for example, be formulated assuppositories e.g. containing conventional suppository bases for use inhuman or veterinary medicine or as pessaries e.g. containingconventional pessary bases.

In one embodiment the formulation is provided as a formulation fortopical administration including inhalation.

Suitable inhalable preparations include inhalable powders, meteringaerosols containing propellant gases or inhalable solutions free frompropellant gases. Inhalable powders according to the disclosurecontaining the active substance may consist solely of the abovementionedactive substances or of a mixture of the abovementioned activesubstances with physiologically acceptable excipient.

These inhalable powders may include monosaccharides (e.g. glucose orarabinose), disaccharides (e.g. lactose, saccharose, maltose), oligo-and polysaccharides (e.g. dextranes), polyalcohols (e.g. sorbitol,mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) ormixtures of these with one another. Mono- or di-saccharides arepreferably used, the use of lactose or glucose, particularly but notexclusively in the form of their hydrates.

Particles for deposition in the lung require a particle size less than10 microns, such as 1-9 microns suitably from 0.1 to 5 μm, particularlypreferably from 1 to 5 μm. The particle size of the active (i.e. thecompound according to the disclosure) should be in this range. The sizeof particle of excipients such as lactose may be larger than this range.

The propellant gases which can be used to prepare the inhalable aerosolsare known from the prior art. Suitable propellant gases are selectedfrom among hydrocarbons such as n-propane, n-butane or isobutane andhalohydrocarbons such as chlorinated and/or fluorinated derivatives ofmethane, ethane, propane, butane, cyclopropane or cyclobutane. Theabove-mentioned propellent gases may be used on their own or in mixturesthereof.

Particularly suitable propellant gases are halogenated alkanederivatives selected from among TG11, TG12, TG 134a and TG227. Of theabovementioned halogenated hydrocarbons, TG134a(1,1,1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are suitable for use in formulations ofthe present invention.

The propellant-gas-containing inhalable aerosols may also contain otheringredients such as co-solvents, stabilisers, surface-active agents(surfactants), antioxidants, lubricants and means for adjusting the pH.All these ingredients are known in the art.

The propellant-gas-containing inhalable aerosols according to theinvention may contain up to 5% by weight of active substance. Aerosolsaccording to the disclosure may contain, for example, 0.002 to 5% byweight, 0.01 to 3% by weight, 0.015 to 2% by weight, 0.1 to 2% byweight, 0.5 to 2% by weight or 0.5 to 1% by weight of active.

The compounds of the disclosure may also be used in combination withother therapeutic agents. The disclosure thus provides, in a furtheraspect, a combination comprising a compound of the present disclosure ora pharmaceutically acceptable derivative thereof together with a furthertherapeutic agent. The combination may be provided as a co-formulationor simply packaged together as separate formulations, for simultaneousor sequential delivery.

A list of possible active ingredients that may complement thetherapeutic activity of the glycodendrimer according to the presentdisclosure is given above.

Therapeutic antibodies may also complement the therapeutic activity ofthe glycodendrimer according to the present disclosure. Examples oftherapeutic antibodies include anti-TNF-alpha antibodies, for exampleetanercept, infliximab, adalimumab, certolizumab pegol, golimumab;Interleukin 1 antibodies, for example anakinra; rituximab; abatacept;and tocilizumab.

It is to be understood that not all of the compounds (or molecules) ofthe combination need be administered by the same route. Thus, if thetherapy comprises more than one active component, then those componentsmay be administered by different routes.

The individual components of such combinations may be administeredeither sequentially or simultaneously in separate or combinedpharmaceutical formulations by any convenient route.

When administration is sequential, either the compound of the disclosureor the second (further) therapeutic agent may be administered first.When administration is simultaneous, the combination may be administeredeither in the same or a different pharmaceutical composition.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalformulations comprising a combination as defined above together with apharmaceutically acceptable carrier or excipient comprise a furtheraspect of the disclosure.

When combined in the same formulation it will be appreciated that thetwo compounds must be stable and compatible with each other and theother components of the formulation. When formulated separately they maybe provided in any convenient formulation, in such manner as are knownfor such compounds in the art.

The compositions may contain from 0.01-99% of the active material. Fortopical administration, for example, the composition will generallycontain from 0.01-10%, more preferably 0.01-1% of the active material.

Processes for preparing said pharmaceutical formulations may, forexample, be performed under controlled environments, such as controlledhumidity conditions.

In one embodiment the pharmaceutical formulation is protected fromlight, for example is stored in amber bottles or vials, foil wrapped orpackaged, such as foil overwrapped or filled into foil blister packs orfoil sachets. In one embodiment the pharmaceutical formulation isprotection from moisture, for example foil wrapped or packaged, such asfoil overwrapped or filled into foil blister packs or foil sachets. Inone embodiment the formulation is protected from air/oxygen, for exampleby storage under nitrogen.

Blister packaging is well known to those skilled in the art, however, inone embodiment the blister is a so-called tropical blister availablefrom amcor or a similar blister available from Alcan. US2006/0283758incorporated by reference discloses certain blister packs suitable foruse with formulations of the invention.

Advantageously appropriately packaged formulations of the presentdisclosure can be stored at room temperature.

When a compound of the disclosure or a pharmaceutically acceptablederivative thereof is used in combination with a second therapeuticagent active against the same disease state, the dose of each compoundmay be the same or differ from that employed when the compound is usedalone. Appropriate doses will be readily appreciated by those skilled inthe art. It will also be appreciated that the amount of a compound ofthe disclosure required for use in treatment will vary with the natureof the condition being treated and the age and the condition of thepatient and will be ultimately at the discretion of the attendantphysician or veterinarian.

Typically, a physician will determine the actual dosage which will bemost suitable for an individual subject. The specific dose level andfrequency of dosage for any particular individual may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, drug combination, the severity ofthe particular condition, and the individual undergoing therapy.

For oral and parenteral administration to humans, the daily dosage levelof the agent may be in single or divided doses. For systemicadministration the daily dose as employed for adult human treatment willrange from 2-100 mg/Kg body weight, preferably 5-60 mg/Kg body weight,which may be administered in 1 to 4 daily doses, for example, dependingon the route of administration and the condition of the patient. Whenthe composition comprises dosage units, each unit will preferablycontain 100 mg to 1 g of active ingredient. The duration of treatmentwill be dictated by the rate of response rather than by arbitrarynumbers of days. In one embodiment the treatment regime is continued for1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21 or more days.

As described above, the compounds of the present disclosure andcomposition comprising the same may be employed in the treatment orprophylaxis of humans and/or animals.

Managing Uncontrolled Immunological Trauma in Elective Surgery

Elective surgery causes the release of tissue enzymes that degrade highmolecular weight hyaluronan into low molecular weight hyaluronan. Thesmall fragments trigger TLR4 mediated pro-inflammatory responses in amanner that is almost identical to bacterially derived LPS. An excessivepro-inflammatory cytokine release interferes with the normal phases ofwound healing. The excessive angiogenesis that accompanies this hostinnate immune response increases pro-inflammatory monocyte recruitmentto the wound site. Scarring is due to a persistent pro-inflammatoryresponse that promotes fibroblast proliferation. Shaunak postulated thatearly inhibition of an immuno-modulatory pathway and an anti-angiogenicpathway would enable physiological (rather than pathological) repair andregeneration of surgically induced injury without causing scar tissueformation [8].

A rabbit model of glaucoma filtration surgery was chosen because thesurgical intervention is precisely defined, and because surgical failureresults from an excessive pro-inflammatory response combined with aneo-angiogenic response. When used in combination, PAMAM dendrimerglucosamine and PAMAM dendrimer glucosamine 6-sulfate increased thesuccess rate of glaucoma filtration surgery from 30% to 80% (P=0.029) inthis clinically validated rabbit model. Therefore, this combination ofdendrimer based drugs safely and synergistically prevented scar tissueformation after surgery. Histological studies showed that the degree oftissue based inflammatory cell infiltration and abnormal collagenformation was minimal [5,18].

It is believed that the glycodendrimers of the present invention alsohave these properties. Thus there is provided a glycodendrimer of thepresent disclosure or a combination thereof or a pharmaceuticalcomposition comprising the same for use in treatment or prophylaxis, inparticular the treatment or prophylaxis of surgery induced tissuesdamage or tissue injury or damage that if untreated would lead toscaring and impairment of the original tissue function, for example forthe treatment or prevention of scar tissue in eye tissue.

Tissue Damaging Cascades

Both surgical trauma and bacterial infections can lead to severe tissueinjury that can be triggered by cell surface TLR4 mediatedreceptor-ligand interactions. These polyvalent interactions betweenbacterially derived ligands as well as endogenous hyaluronan fragmentscan lead to the release of life threatening pro-inflammatory cytokinessuch as IL-6 and TNF-alpha. As a result, this pathway is tightlyregulated in all biological organisms. The checkpoints that initiate aswell as arrest this tissue damaging cascade are important because theyhave the potential of being manipulated with pharmaceutical drugs.

Thus in one embodiment there is provided use of a compound according tothe disclosure and compositions comprising the same for the treatment orprophylaxis of scarring, including excessive scarring, particularlyafter surgery, whether internal to the body or relating to a surfaceorgan of the body; e.g. such as the skin or a mucosal surface or asurface related to the eye.

The Inflammatory Response Associated With Bacterial Infections

Fundamental to innate immunity are the pattern recognition receptors(TLRs) that recognize pathogen associated molecular patterns. They allowthe immune system to distinguish self structures from pathogenassociated non self molecules. They are the first line of host defenseagainst invading pathogens [23].

TLR4 on macrophages and dendritic cells is the key cell surfacereceptor. Antigen mediated triggering leads to cytokine expression,dendritic cell maturation, and adaptive immune responses.

Only a very short stimulation of TLR4 is required to lead to dendriticcell maturation and T cell stimulation. This contrasts with theprolonged and sustained stimulation of TLR4 that is required for theinduction of pro-inflammatory cytokines such as TNF-α and IL-6. Distinctthresholds therefore exist within the TLR4-MD2-LPS complex (at the levelof the cell surface) for inducing the expression of CD markers ofcellular differentiation compared to the release of cytokines [1]. Thisunique nature of TLR4 compared to all other TLR receptors has only beenrecently recognized [24].

Thus in one aspect there is provided use of compounds of the presentdisclosure and compositions comprising same for the treatment orprophylaxis of inflammatory responses or inflammatory disease, forexample a response mediated by increased levels of one or more cytokinesselected from the group comprising IL-6, TNF-alpha, IL-8, IL-1 beta andMIP-1 beta. In one embodiment the inflammatory mechanism is in response,for example in response to LPS and/or hyaluronan fragments that bind tothe cell surface receptor TLR4 and/or to bacterial infection, forexample in the lining of the gut.

In one embodiment there is provided glycodendrimer, population orformulation according to the present disclosure for use in the treatmentor prophylaxis of a disease that is associated with an excessivepro-inflammatory cytokine response by the host/patient.

In one embodiment there is provided a glycodendrimer or populationaccording to the present disclosure or a composition comprising the samefor the treatment or prophylaxis of inflammation associated with Gramnegative infections, for example Gram negative infection is associatedwith diarrhoea, such as those caused by Shigella sp. and Salmonella sp.

In one embodiment there is provided a glycodendrimer, population orformulation according to the present disclosure wherein the infection iscaused by Escherichia coli, Klebsiella aeruginosa, Staphylococcusaureus, Escherichia faecalis, Pseudomonas aerugenosa, and/or any otherinfectious organism.

In one embodiment there is provided a glycodendrimer according to thepresent disclosure or a composition comprising the same for thetreatment or prophylaxis of inflammation associated with Gram negativeinfections, for example Gram negative infection is associated withinflammatory diarrhoeas, such as those caused by Shigella sp.,Salmonella sp., Campylobacter sp., Clostridium difficile and E. coli.

In one embodiment there is provided a glycodendrimer or populationaccording to the present disclosure or a composition comprising the samefor use in the treatment or prophylaxis of inflammatory bowel disease,such as Crohn's Disease and/or Ulcerative Colitis.

In one embodiment there is provided a glycodendrimer according to thepresent disclosure or a composition comprising the same for use in thetreatment or prophylaxis of those forms of irritable bowel disease, forexample associated with an excessive stimulation of Toll Like receptorsby gut bacteria.

In one embodiment there is provided a glycodendrimer according to thepresent disclosure or a composition comprising the same for use in thetreatment or prophylaxis of abnormally excessive host pro-inflammatorycytokine mediated responses in the respiratory system such as those thatoccur in allergy, asthma or after a bacterial and/or viral infection.

In one embodiment there is provided a glycodendrimer, population orformulation according to the present disclosure for use in the treatmentor prophylaxis of inflammatory respiratory responses, such as allergyand/or asthma.

In one embodiment there is provided a glycodendrimer or populationaccording to the present disclosure or a composition comprising the samefor use in the treatment or prophylaxis of excessive scarring duringwound healing, keloid formation, eczema and psoriasis.

In one embodiment there is provided a glycodendrimer or populationaccording to the present disclosure or a composition comprising the samefor use in the treatment or prophylaxis of transplants or organs ortissue, such as corneal and/or skin transplantation.

In one embodiment there is provided a glycodendrimer according to thepresent disclosure, for use in the treatment or prevent of undesirableangiogenesis or restenosis (for example after insertion of a stent).

In one embodiment there is provided a stent coated with a compoundaccording to the present disclosure.

In one embodiment there is provided a glycodendrimer or populationaccording to the present disclosure or a composition comprising the samefor use in the treatment or prophylaxis of gingivitis.

In one embodiment there is provided a glycodendrimer according to thepresent disclosure or a composition comprising the same for use in thetreatment or prophylaxis of rheumatoid arthritis or osteoporosis.

In one embodiment glycodendrimers according to the present disclosure orpharmaceutical formulations thereof are suitable for administrationdirectly to the eye as eye drops, by deposition of a pellet in or aroundthe eye, or by injection into any chamber within the eye, or by directinfusion through an organ, for example at a concentration ranging from2.5 to 2,500 μg/ml.

In one embodiment there is provided a method of treatment comprisingadministering a therapeutically effective amount of a glycodendrimer orpopulation according to the present disclosure or a compositioncomprising same to a patient in need thereof, in particular fortreatment or prophylaxis of an indication described herein.

In one embodiment there is provided use of a glycodendrimer orcomposition comprising the same for the manufacture of a medicament forthe treatment of an indication described herein.

Process Chemistry For Zero Length Amide Bond Formation

The detailed divergent chemical synthesis of polypropyletheriminedendrimers cores up to generation 3 (i.e., with 16 peripheral carboxylicacid groups) has been described in detail in [19].

Starting with an oxygen core, the dendrimer was synthesised byrepetitive cycles consisting of two reductions and two Michael additionreactions. These repetitive and consecutive reactions were performedusing alpha-beta-unsaturated ester and nitrile as monomers, andsupported metal catalysts and metal hydrides as reagents. Esters areconverted to alcohols followed by conversion of alcohols to ethers withpendant nitriles, followed by conversion of nitriles to primary amines,followed by conversion of primary amines to tertiary amines with pendantesters. The procedure described is long but simple and the yield isgood.

Chromatography after the sequential synthesis of each dendrimergeneration by HPLC and/or column chromatograph can be used to ensurethat the single entity generation 3 polypropyletherimine is obtained asa final product. A MALDI-MS of this molecule is shown in thesupplementary files attached to the following paper [19].

Synthesis of large generation polypropyletherimine dendrimers isdescribed in [22].

A process to covalently link a biologically inactive glucosaminemolecule to a biologically inactive anionic carboxylic acid terminateddendrimer molecule wherein the dendrimer core was reacted with the sugarmolecule such as the glucosamine molecule in the presence of a couplingagent such as carbodiimide coupling or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride {EDC}. The reaction is carried out in anaqueous solvent, for example water and at room temperature and withoutan exogenous heat source. An analogous process is described for examplein WO 03/089010. In one embodiment the core is a 3, 3.5 or 4 generationdendrimer.

This process had the advantage of comprising a single synthetic step tocreate a covalent zero length amide bond, using water as the solvent,and it can be performed at room temperature (i.e., 18-26° C.). Thisprocess also has the advantage that it avoided the need for the use oforganic solvents that are often toxic in vivo.

Furthermore, organic solvents require additional, complicated andexpensive purification procedures for isolating the final product fromthe organic solvent. Conjugation, that is to say, covalent linkage ofthe components in an aqueous environment facilitates the simple andstraightforward purification of the final medicinal product. This hasimportant industrial advantages, and manufacturing advantages, andregulatory advantages for a new pharmaceutical drug.

Suitably, the dendrimers cores are covalently linked to compoundscontaining amino groups, for example, amine groups, for example, primaryamine groups, such as amino sugars in particular glucosamine.

Typically the covalent link formed by the conjugation is stable over aperiod of more than 18 months, which may be important in the shelf-lifeof a pharmaceutical product. In one embodiment the glycodendrimer formedis lyophilised. This may further extend the shelf life of the molecule.

Thus in one aspect there is provided a process preparing aglycodendrimer according to the present invention comprising the step ofconjugating a glucosamine molecule to a dendrimer core, in particular apolypropyletherimine dendrimer core, for example prepared divergently,such as a generation 3 core. In one embodiment, a covalent zero lengthamide bond is formed between the sugar and a carboxylic acid residue onthe core. In one embodiment a coupling agent is employed selected from1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and EDC.

In one embodiment the process employs water as the solvent. In oneembodiment the reaction is carried out at less than 40° C. without theapplication of an external, additional energy source.

In one embodiment the glycodendrimer is purified after conjugation ofthe core to the sugar, for example purification may be effected bydialysis and/or by column chromatograghy.

In one embodiment the glycodendrimer obtained from said process is amonodispersion, for example with 2 glucosamine molecules attached atopposite ends of the surface of the dendrimer.

In one embodiment the disclosure relates to products obtainable fromsaid process.

It is important to note that these anionic carboxylic acid terminateddendrimers (without glucosamine attached) have no effect onpro-inflammatory cytokines such as IL-6, IL-1 beta, TNF-alpha and MIP-1beta even at very high doses. It is also important to note thatglucosamine (on its own) also has no effect on pro-inflammatorycytokines such as IL-6, IL-1 beta, TNF-alpha and MIP-1 beta even at highdoses.

Nevertheless, the present disclosure further provides use of thedendrimer cores (polypropyletherimine cores, particularly generation 3cores), described herein in the preparation of a glycodendrimer, inparticular a therapeutic glycodendrimer, in particular as describedherein.

In one aspect there is provided use of glucosamine, in particular asdescribed herein for the preparation of a glycodendrimer, in particulara therapeutic glycodendrimer, in particular as described herein.

Average as employed herein is intended to refer to a mean average or amodal average.

All citation and documents referred herein are specifically incorporatedby reference.

Comprising in the context of the present invention means including.

Described above are embodiments comprising certain integers. Embodimentsof the invention described above can be combined as technicallyappropriate. The present disclosure also extends to correspondingembodiments consisting of said integers as herein described.

All references to literature and patent documents are incorporated byreference.

EXAMPLES

The table below describes some of the compounds prepared over severalyears in the search for a biologically active dendrimer. Table 1 is asummary of the glycodendrimers prepared and analysed

Biological Activity (reduction of pro- GLYCODENDRIMER inflammatory CoreGeneration Sugar cytokines) Triazine 2 Glucosamine none (6 equivalentsused) Triazine 2 Glucosamine none (12 equivalents used) Triazine 2Glucosamine none (120 equivalents used) Triazine 2 Glucosamine none (120equivalents used at pH 5) Triazine 3 Glucosamine none 50% loadingTriazine 3 Glucosamine none 58% loading Triazine 3 Glucosamine nonePAMAM hybrid 0.5 12.5% loading Triazine 3 Glucosamine yes PAMAM hybrid1.5 18.75% loading (but a mixture of multiple species of molecules thatare not all closely related)

Example 1 Studies of Generation 3 Polypropyletherimine Carboxylic AcidTerminated Dendrimer Glucosamine Example 1A Synthesis 1

Despite the molecular modelling studies showing that the generation 3polypropyletherimine carboxylic acid terminated dendrimer glucosamine(12.5% loading) would not be biologically active, we neverthelessproceeded with its chemical synthesis and its biological testing.Divergent synthesis of a generation 3 carboxylic acid terminatedpolypropyletherimine dendrimer has been previously described by—[19].

50 mg of a monodispersed generation 3 polypropyletherimine dendrimer(MWt 2,667) was dissolved in 0.5 ml water. 50 mg of glucosamine wasdissolved in 1 mg/ml water, added to the dendrimer, and the pH adjustedto pH 5. EDC (208 mg) was dissolved in 4.1 ml of water and added to themixture, and the pH adjusted to 5.0. The reaction was stirred for 3 h atroom temperature with constant monitoring of the pH which was adjustedto pH 5.0. After 3 h, the reaction was transferred to a 2,000 MWtcut-off dialysis cassette and then dialysed for 24 h with 3 changes ofwater. All of the water used in the synthesis reactions and in thedialysis was endotoxin free. The dialysed reactions were lyophilised andresuspended in water at 50 mg/ml, and confirmed to be endotoxin free at<0.1 EU/ml using the limulus assay.

MALDI-MS and NMR studies showed that the monodispersed generation 3polypropyletherimine dendrimer (with 16 peripheral carboxylic acidgroups) had a loading of 2 glucosamine molecules per dendrimer (actualMWt 3,061; theoretical calculated weight 3,061). This gave a percentageglucosamine loading of 12.5%; i.e. 2 glucosamine molecules on the 16peripheral carboxylic acid groups of this dendrimer.

Example 1B (Alternative Process)

To prepare endotoxin-free solutions and glassware, all glassware andmagnetic fleas were autoclaved twice at 123° C. for 15 minutes. Thesynthesis was performed using sterile and endotoxin free disposableplastic tissue culture grade 50 ml universal tubes. All other disposableplastic pipettes, universals and syringes are certified endotoxin-free(i.e., endotoxin <0.01 endotoxin units (EU)/ml). Endotoxin free waterfor injection was used. This means that endotoxin contamination wasreduced to a minimum from the very start of the partial glycosylationsynthesis reaction. In order to eliminate any bacterial contamination ofthe pH probe used, it was first immersed in 1 N HCl solution for 15minutes before being used, and then washed 4 times with endotoxin freewater to remove any residual acid.

150 mg of the Generation 3 polypropyletherimine dendrimer was dissolvedin 1.5 ml sterile endotoxin free water to give a concentration of 100mg/ml. A magnetic flea was put into the 50 ml Falcon tube and stirred ata slow speed on a magnetic stirring plate. 150 mg of D-glucosaminehydrochloride from Sigma UK (catalogue number G4875) was dissolved in 3ml of sterile water to give a concentration of 50 mg/ml. It was thenadded to the dissolved dendrimer. This was equivalent to 0.78glucosamine molecules per peripheral carboxylic acid group on thedendrimer. The pH of the resulting solution (which is quite acidic) wasreadjusted to 5.0 using 1 N NaOH. Then, 334 mg of1-ethyl-3-3-dimethylaminopropyl carboiimide hydrochloride (EDCI, SigmaUK) was dissolved in 6.7 ml sterile water to give a concentration of 50mg/ml. This was equivalent to 1.94 EDCI molecules per peripheralcarboxylic acid group on the dendrimer. The EDCI solution was then addedimmediately to the solution containing the dendrimer and the glucosamineThe pH was readjusted to 5.0 with 1 N HCl. The pH of the reactionsolution was readjusted to 5.0 by adding 1 N HCl every 15-30 minutesduring the 3 hour reaction. The final volume of the reaction was about12-12.5 ml.

For the dialysis steps that followed, all glass beakers and magneticstir bars were autoclaved twice at 123° C. for 15 minutes. Endotoxinfree water (Baxter Healthcare) was used. One of two procedures wasfollowed. The first procedure used a Float-a-Lyzer dialysis cassette(Spectrapor) with a MWt cut-off of 1 kDa and a volume of 10 ml. In thiscase, the dialysis tube was rehydrated using endotoxin-free water,filled with the solution, and then dialysed (with stirring) for 1 hourin 1 L of endotoxin free water at 4° C. All subsequent dialysis was at4° C. The water was then replaced and the dialysis continued overnight.The following day, the water was replaced and the dialysis continued foranother 24 hours; i.e., a further 4 changes of water at 3.5 h intervals,and including another overnight dialysis. In total, this meant 42 hoursof dialysis at 4° C. with 7 changes of water. In the second dialysisprocedure, a Slide-a-lyser dialysis cassette (Pierce) with a MWt cut-offof 2 kDa and a volume of 3-12 ml was used. The dialysis cassette wasrehydrated using endotoxin-free water, filled with the solution, andthen dialysed (with stirring) for 1 hour in 1 L of endotoxin free waterat 4° C. All subsequent dialysis was at 4° C. The water was thenreplaced and the dialysis continued overnight. The following day, thewater was replaced and the dialysis continued for another 24 hours, witha further 4 changes of water at 3.5 hour intervals, and includinganother overnight dialysis. In total, this meant 42 hours of dialysis at4° C. with 7 changes of water. The dialysate was then removed from thecassettes with a needle and syringe and filtered through 0.2 μm sterilefilters and placed in pre-weighed sterile 50 ml Falcon tubes. It wasthen frozen for at least 1 hour at −80° C. Parafilm was placed over themouth of the 50 ml tube that contained the frozen dendrimer glucosamineand pierced with a needle. The tube was then placed in a freeze drier(which has been pre-run for 30 min) and its contents left to lyophilizefor 48 hours.

The lyophilised product was confirmed to be endotoxin free at <0.1 EU/mlusing the limulus amoebocyte assay. As the dendrimer glucosamine ishygroscopic, it was stored in small airtight containers, and underargon, and at 4° C., and wrapped in aluminium foil. H-NMR and C-NMR andMALDI-MS studies showed that the Generation 3 polypropyletheriminedendrimer (with 16 peripheral carboxylic acid groups) had a loading of 2glucosamine molecules per dendrimer. This gave a percentage glucosamineloading of 12.5%; i.e., 2 glucosamine molecules on the 16 peripheralcarboxylic acid groups of this dendrimer. In addition, there was noresidual small molecule contamination of the product with acrylonitrile,acrylic acid, free glucosamine or urea.

Example 2 Biological Studies With the Generation 3 PolypropyletherimineCarboxylic Acid Terminated Dendrimer Glucosamine Example 2A

Cellular cytotoxicity was determined as follows. Peripheral bloodmononuclear cells were isolated from fresh human blood by densitygradient centrifugation and re-suspended in growth medium (RPMI 1640, 20mM L-glutamine, penicillin [250 IU/ml], streptomycin [250 μg/m1] and 10%endotoxin free human serum). They were allowed to adhere to plastictissue culture plates for 1 h. The plates were washed, the adherentmonocytes scraped with a cell scraper, and the cell density adjusted to10⁶ cells/ml. 200 μL of these monocytes were plated in a 96 well plateat a density of 10⁶ cells/ml. The generation 3 polypropyletheriminedendrimer (0 to 400 μg/ml) was added to monocytes and incubated for 24h. Cell viability was assessed using the MTT assay. No cytotoxic effectof the generation 3 polypropyletherimine carboxylic acid terminateddendrimer was found up to the highest concentration tested. Thegeneration 3 polypropyletherimine dendrimer glucosamine (0 to 400 μL/ml)was then added to monocytes and incubated for 24 h. Cell viability wasassessed using the MTT assay. No cytotoxic effect of the generation 3polypropyletherimine carboxylic acid terminated dendrimer glucosaminewas found up to the highest concentration tested (FIG. 7).

Example 2B The Ability of the Generation 3 PolypropyletherimineDendrimer Glucosamine to Reduce Pro-Inflammatory Cytokine Production wasDetermined in a Human Monocyte/Macrophage Based Assay With LPS BeingUsed to Stimulate the Release of Pro-Inflammatory Cytokines

1 ml aliquots of human monocytes (10⁶ cells/ml) were transferred to a24-well tissue culture plate and incubated for 30 min at 37° C. To theseadherent monocytes, endotoxin free (i.e., <0.01 EU/ml) generation 3polypropyletherimine carboxylic acid terminated dendrimer glucosaminewas added at concentrations from 50 to 200 μg/ml and incubated for 1 hat 37° C. LPS (Salmonella Minnesota, Sigma. Catalogue number L9764) wasadded at 25 ng/ml. Positive controls were cells treated with LPS only,and the negative controls were untreated cells, or cells incubated withthe dendrimer only. The cells were then maintained at 37° C. with 5% CO₂for 3 h. Media was then removed, cells lysed in 500 μL of Tri-reagent(Sigma) and RNA extracted. Reverse transcription was performed using aQiagen RT kit. Aliquots of cDNA were then subjected to quantitativereal-time PCR for a panel of cytokines. A large reduction in thesynthesis of the pro-inflammatory cytokines IL-6, TNF-alpha, IL-8, andMIP-1 beta was seen in the presence of the generation 3 anionicpolypropyletherimine carboxylic acid terminated dendrimer glucosamine ata concentration of 100 μg/ml. In addition, no change was seen in theanti-inflammatory cytokines IL-10 and interferon-beta (FIGS. 7 and 8).This was a very surprising and unexpected biological result.

Example 2C Detailed Biological Studies with the Generation 3Polypropyletherimine Carboxylic Acid Terminated Dendrimer Glucosamine

The reduction in the production of the pro-inflammatory cytokines IL-6,TNF-alpha, IL-8 and MIP-1 beta seen with generation 3polypropyletherimine carboxylic acid terminated dendrimer glucosamine ata dose of 100 μg/ml with Salmonella sp. LPS was a surprising resultbecause all of our molecular modelling studies had suggested that thegeneration 3 polypropyletherimine dendrimer glucosamine (with only 16carboxylic acids available for the conjugation to glucosamine)would:—(1) be too small a molecule; (2) it would not have the correctphysic-chemical characteristics to act as an effective antagonist of theMD2-TLR4-LPS mediated pro-inflammatory cytokine response.

To further verify the validity of the biological results obtained abovewith the generation 3 polypropyletherimine carboxylic acid terminateddendrimer glucosamine, additional biological experiments were performed.

Example 2D Assay for Inhibition of Pro-Inflammatory Cytokine Productionby Generation 3 Polypropyletherimine Carboxylic Acid TerminatedDendrimer Glucosamine After Challenge With Shigella Sp. Wild Type LPSAnd Molecularly Modified Waal Mutant Shigella Sp. LPS

Wild type M90 and the Shigella mutant (waaL—which has no O-antigenglucosylation pattern—see FIG. 9 and also [25]) were propagated andtheir LPS extracted by phenol extraction. The LPS derived from:—(1)wild-type Shigella M90; (2) waaL, were added at 25 ng/ml.

Treatment of human monocytes with LPS isolated from wild type and waaLShigella mutants induced the expression of the pro-inflammatorycytokines IL-6, TNF-alpha, IL-8 and MIP-1 beta at similar levels.Pre-treatment of monocytes with 25 to 100 μg/ml of generation 3 anionicpolypropyletherimine carboxylic acid terminated dendrimer glucosamineresulted in a dose dependant reduction of all of these pro-inflammatorycytokines when Shigella wild type LPS was used (FIG. 10), and also whenShigella waaL mutant LPS was also used (FIG. 10). A modest reduction ofthe pro-inflammatory cytokines was seen with 25 μg/ml of generation 3polypropyletherimine carboxylic acid terminated dendrimer glucosaminewith a maximal effect seen at a dose of 100 μg/ml of generation 3anionic polypropyletherimine carboxylic acid terminated dendrimerglucosamine.

Example 2E Assay for Inhibition of Pro-Inflammatory Cytokine Productionby G3.5 Polyamidoamine Dendrimer Glucosamine and G3 PolypropyletherimineDendrimer Glucosamine After Challenge of Rabbits Infected With Wild TypeShigella Sp. Infection

Surgery to create ileal loop sacs in rabbits was performed as describedpreviously [26]. This rabbit model has proven invaluable for studies ofmucosal inflammation and bacterial invasion in infectious diarrheas.Shigellosis (and the typhoid fever of salmonella) lead to inflammatorychanges in the epithelium associated lymphoid follicles; i.e., Peyer'spatches [26,27]. The localised but excessive IL-6 and TNF-alpha mediatedpro-inflammatory cytokine response that follows leads to the destructionof the intestinal epithelium because:—(1) the organism multiples in thelumen of the isolated ileal loops; (2) a severe host mediatedpro-inflammatory response occurs; (3) the mucosal barrier is damaged;and (4) bacterial invasion occurs through the gut associated lymphoidtissues (i.e., Peyer's patches). This is associated with an infiltrationof:—(a) blood derived monocytes that differentiate into macrophages; and(b) neutrophils. The inflammatory changes in this rabbit based ilealloop model depend directly upon the presence of shigella LPS and theproduction of IL-6 and TNF-alpha by the large numbers of incoming bloodmonocytes.

For these experiments, wild-type M90 Shigella flexneri bacteria weregrown in broth, harvested whilst still in the exponential phase ofgrowth, and diluted to give a concentration of 2×10⁷ bacteria per ml.The rabbit ileal loops containing Peyer's patches were treated with 2.5mg of generation 3.5 polyamidoamine (PAMAM) dendrimer glucosamine andthe control loop was treated with 2.5 mg of generation 3.5polyamidoamine (PAMAM) dendrimer. These molecules were administered in avolume of 1 ml of water. 10⁷ M90 Shigella flexneri bacteria were theninjected into each ileal loop. The abdomen was closed and the rabbitsleft for 16 h. The rabbits were then killed and the ileal loops andPeyer's patches sampled. Tissue was immediately immersed in 2 ml ofTriReagent, homogenised for 1 min using a Polytron homogenizer and theRNA extracted for quantitative real-time RT-PCR based studies ofpro-inflammatory cytokines.

No adverse reaction were seen when dendrimer glucosamine wasadministered into the ileal loops. In these experiments, and using wildtype Shigella flexneri (M9OT strain; 10⁷ organisms/loop), it was shownthat:—(1) Neither the dendrimer nor the PAMAM dendrimer glucosamine hadan anti-microbial effect on the Shigella flexneri when administered intothe ileal loops; (2) A significant and therapeutically beneficial effectcould be achieved after the oral administration of dendrimer glucosamineinto the gut lumen; (3) No clinical toxicity was seen; (4) Functionaltissue injury was reduced by PAMAM dendrimer glucosamine as determinedby a 90% reduction in the volume of bloody diarrhoea that occurs duringa shigella infection; (5) Histo-pathological studies showed thatinfected animals receiving dendrimer only have a marked tissuepro-inflammatory cell infiltrate in the gut wall and gut lumen, edema ofthe lymphoid follicles (i.e., Peyer's patches), extensive destruction ofthe epithelium associated with the lymphoid follicle, and inflammation,hemorrhagic infiltration, dilatation, shortening and destruction of thevilli between the lymphoid follicles (i.e., Peyer's patches). Incontrast, the histo-pathological findings were quite different in theinfected animals treated by the oral administration of dendrimerglucosamine into the gut lumen in that there were minimal morphologicalchanges in the mucosal villi and Peyer's patch follicles, and the tissuebased inflammatory cell infiltrate present did not cause epitheliumrupture ; (6) There was a large reduction in pro-inflammatory cytokineproduction as demonstrated by a 1,000-fold reduction in IL-6 production,a 100-fold reduction in IL-8 production, and a 100-fold reduction inTNF-alpha production. Importantly, for the anti-inflammatory cytokineIL-10, there was no difference. For the dendritic cell maturationmediator—Interferon-beta—there was also no difference. For theepithelial cell anti-microbial peptide β-defensin, there was nodifference. These animal model based results showed that dendrimerglucosamine can substantially alter the course of a gut infection evenwhen an antibiotic is not co-administered during the course of theseexperiments.

The above experiment was repeated with a G3 polypropyletheriminedendrimer glucosamine according to the present invention.

Example 2F Assay for Inhibition of pro-Inflammatory Cytokine Productionby Generation 3 Polypropyletherimine Carboxylic Acid TerminatedDendrimer Glucosamine After Challenge With LPS, Live E. Coli Bacteria,Live Klebsiella Aeruginosa Bacteria, Live S. Aureus Bacteria, Live E.Faecalis Bacteria, and Live Pseudomonas Aerugenosa Bacteria

1 ml aliquots of human monocytes (10⁶ cells/nil) were transferred to a24-well tissue culture plate and incubated for 30 min at 37° C. To theseadherent monocytes, endotoxin free dendrimer glucosamine was added atconcentrations from 50 to 200 μg/ml and incubated for 1 h at 37° C. LiveEscherichia coli, Klebsiella aeruginosa, Staphylococcus aureus,Escherichia faecalis, and Pseudomonas aerugenosa from overnightbacterial cultures were added to separate plates of monocytes at amultiplicity of infection of 10 infectious bacteria per monocyte (totalvolume 50 μL) and centrifuged at 780 g for 7 min to maximise the contactbetween the bacteria and the monocytes. The cells were then maintainedat 37° C. with 5% CO₂ for 1 h. Gentamicin (100 μg/m1) was then added andthe tissue culture plate incubated for an additional 2 h. Media was thenremoved, cells lysed in 500 μL Tri-reagent (Sigma), and the RNAextracted. Reverse transcription was performed using a Qiagen RT kit.Aliquots of cDNA were then subjected to quantitative real-time mRNA PCRfor pro-inflammatory cytokines.

A large reduction in the synthesis of the pro-inflammatory cytokinesIL-6 (FIG. 12), TNF-alpha (FIG. 13) and MIP-1 beta (FIG. 14) was seen inthe presence of the generation 3 polypropyletherimine carboxylic acidterminated dendrimer glucosamine (12.5% loading) molecule at aconcentration of 100 μg/ml.

FIG. 1: Illustration showing competition for cell surface Toll-likereceptor 4 (TLR4) between the agonist (lipopolysaccharide [LPS])) andthe antagonist (dendrimer glucosamine). MD2 is a protein and R1, R2, R3,and R4 are acyl chains.

FIG. 2: shows a diagrammatic representation of a PAMAM glucosaminedendrimer.

FIG. 3: Generation 3 anionic carboxylic acid terminated (i.e., 16peripheral carboxylic acid groups) polypropyletherimine dendrimer core.The entire and symmetrical dendrimer is shown in 2-dimensions. Thisdendrimer does not have internal cavities. It is therefore not suitablefor acting as a drug delivery dendrimer.

FIG. 4: Generation 3 anionic carboxylic acid terminated (i.e., 16peripheral carboxylic acid groups) polypropyletherimine dendrimerglucosamine with a 12.5% surface loading of glucosamine (i.e., 2glucosamine molecules) with a zero length amide bond between thedendrimer and the glucosamine The glucosamine molecules are evenlyspaced on the surface of this symmetrical dendrimer as illustrated byeach of the black stars. Each of the arcs (n=2) represents the eightcarboxylic acid groups to one of which is covalently attached aglucosamine molecule. This analytical chemistry observation isconsistent with higher occupied molecular orbital and lowest occupiedmolecular orbital calculations that were performed using the frontiermolecular orbital theory. In the case of a generation 3 anioniccarboxylic acid terminated (i.e., 16 peripheral carboxylic acid groups)polypropyletherimine dendrimer, the incremental addition of eachglucosamine proceeds in a energy favourable manner until 2 glucosaminemolecules have been attached to 2 of the 16 peripheral carboxylic acidgroups available. Thereafter, the higher occupied molecular orbitalenergy values rapidly becomes less favourable to the addition of anyfurther glucosamine molecules. This suggests that the divergent approachto the synthesis of dendrimer glucosamine leads to the favourableaddition of 2 glucosamine molecules on the 16 carboxylic acid groups ofa generation 3 anionic carboxylic acid terminated polypropyletheriminedendrimer. This equates to one glucosamine molecule per eight peripheralcarboxylic acid groups.

FIG. 5: Generation 3 anionic carboxylic acid terminatedpolypropyletherimine dendrimer glucosamine. This figure shows itsoverall molecular surface. This dendrimer does not have internalcavities. It is therefore not suitable for drug delivery purposes.

FIG. 6: Generation 3 anionic carboxylic acid terminatedpolypropyletherimine dendrimer glucosamine This dendrimer glucosaminedoes not have internal cavities and is therefore not suitable for drugdelivery purposes. This figure shows modeling of its hydrophilicsurfaces.

FIG. 7: Cellular cytotoxicity was determined by an MTT assay performedon 10⁵ human monocytes in 96 well plates using 0 to 400 μg/ml of anendotoxin free generation 3 polypropyletherimine anionic carboxylic acidterminated dendrimer glucosamine No cytotoxic effect was observed. Thebiological effect of generation 3 polypropyletherimine anioniccarboxylic acid terminated dendrimer glucosamine was determined in 24well plates using 10⁶ peripheral blood mononuclear cells pretreated with12.5 to 100 μg/ml endotoxin free dendrimer glucosamine for 1 h followedby challenge with 25 ng/ml Salmonella LPS. After 3 h, RNA was extractedand real-time RT PCR performed. When compared with the LPS control,generation 3 polypropyletherimine anionic carboxylic acid terminateddendrimer glucosamine lead to the following reductions inpro-inflammatory cytokines:—an 75-fold reduction in IL-6, a 390-foldreduction in TNF-alpha, a 75-fold reduction in IL-8, and a 165-foldreduction in MIP-1 beta at 100 μg/ml. In contrast, there was no changein the anti-inflammatory cytokines IL-10 and interferon-beta.

FIG. 8: Cellular cytotoxicity was determined by an MTT assay performedon 10⁵ human monocytes in 96 well plates using 0 to 400 μg/ml of anendotoxin free generation 3 polypropyletherimine anionic carboxylic acidterminated dendrimer glucosamine No cytotoxic effect was observed. Thebiological effect of generation 3 polypropyletherimine anioniccarboxylic acid terminated dendrimer glucosamine was determined in 24well plates using 10⁶ monocytes pretreated with 50 to 200 μg/mlendotoxin free dendrimer glucosamine for 1 h followed by challenge with25 ng/ml Salmonella LPS. After 3 h, RNA was extracted and real-time RTPCR performed. When compared with the LPS control, generation 3polypropyletherimine anionic carboxylic acid terminated dendrimerglucosamine lead to the following reductions in pro-inflammatorycytokines:—an 300-fold reduction in IL-6, a 135-fold reduction inTNF-alpha, a 5-fold reduction in IL-8, and a 100-fold reduction in MIP-1beta at 100 μg/ml. In contrast, there was no change in theanti-inflammatory cytokines IL-10 and interferon-beta.

FIG. 9: Schematic representation of the possible truncation mutants ofShigella LPS. M90 is the wild type Shigella flexneri. gtrA is a mutantwith reduced glucosylation. cld (chain length determinant) and dB areO-antigen truncated mutants. waal mutants only have the Lipid A and coresugars without the O-antigen.

FIG. 10: The biological effect of generation 3 polypropyletherimineanionic carboxylic acid terminated dendrimer glucosamine was determinedin 24 well plates using 10⁶ human monocytes pretreated with 25 to 200μg/ml endotoxin free dendrimer glucosamine for 1 h followed by challengewith 25 ng/ml Shigella LPS. After 3 h, RNA was extracted and real-timeRT PCR performed. When compared with the LPS control, generation 3polypropyletherimine anionic carboxylic acid terminated dendrimerglucosamine lead to the following reductions in pro-inflammatorycytokines:—an 30-fold reduction in IL-6, a 4-fold reduction inTNF-alpha, a 15-fold reduction in IL-8, and a 6-fold reduction in MIP-1beta at 100 μg/ml. In contrast, there was no change in theanti-inflammatory cytokines IL-10 and interferon-beta.

FIG. 11: The biological effect of generation 3 polypropyletherimineanionic carboxylic acid terminated dendrimer glucosamine was determinedin 24 well plates using 10⁶ human monocytes pretreated with 25 to 200μg/ml endotoxin free dendrimer glucosamine for 1 h followed by challengewith 25 ng/ml Shigella waaL LPS. After 3 h, RNA was extracted andreal-time RT PCR performed. When compared with the LPS control,generation 3 polypropyletherimine anionic carboxylic acid terminateddendrimer glucosamine lead to the following reductions inpro-inflammatory cytokines:—a 12-fold reduction in IL-6, a 3-foldreduction in TNF-alpha, a 4-fold reduction in IL-8, and a 5-foldreduction in MIP-1 beta at 200 μg/ml. In contrast, there was no changein the anti-inflammatory cytokines IL-10 and interferon-beta.

FIG. 12: The biological effect of generation 3 polypropyletherimineanionic carboxylic acid terminated dendrimer glucosamine was determinedin 24 well plates using 10⁶ human monocytes pretreated with 12.5 to 100μg/m1 endotoxin free dendrimer glucosamine for 1 h followed by challengewith:—(1) 25 ng/ml Salmonella LPS; (2) live E. coli bacteria(multiplicity of infection (MOI)=10); (3) live Pseudomonas aeruginosabacteria (MOI=10); (4) live Klebsiella pneumonia bacteria (MOI=10); (5)live S. aureus bacteria (MOI=10); (6) live E. faecalis bacteria (MOI=10). After a 1 h incubation, gentamycin (100 μg/ml) was added to themonocyte cultures containing bacteria and the incubation was thencontinued for another 2 h, making a total of a 3 hour incubation. TheRNA was then extracted and real-time RT PCR performed. In the case ofevery single stimulant used, generation 3 polypropyletherimine anioniccarboxylic acid terminated dendrimer glucosamine lead to a significantreduction in the pro-inflammatory cytokines IL-6, TNF-alpha, and IL-8.

This figure summarises the results for IL-6. They show the followingreductions:—(1) Salmonella LPS—a 925-fold reduction at 100 μg/ml; (2)live E. coli bacteria—a 30-fold reduction at 100 μg/ml; (3) livePseudomonas aerugenosa bacteria—a 23-fold reduction at 100 μg/ml; (4)live Klebsiella pneumonia bacteria—a 48-fold reduction at 200 μg/ml; (5)live S. aureus bacteria—a 17-fold reduction at 100 μg/ml; (6) live E.faecalis bacteria—a 60-fold reduction at 200 μg/ml.

FIG. 13: The biological effect of generation 3 polypropyletherimineanionic carboxylic acid terminated dendrimer glucosamine was determinedin 24 well plates using 10⁶ human monocytes pretreated with 12.5 to 100μg/ml endotoxin free dendrimer glucosamine for 1 h followed by challengewith:—(1) 25 μg/ml Salmonella LPS; (2) live E. coli bacteria(multiplicity of infection (MOI)=10); (3) live Pseudomonas aeruginosabacteria (MOI=10); (4) live Klebsiella pneumonia bacteria (MOI=10); (5)live S. aureus bacteria (MOI=10); (6) live E. faecalis bacteria(MOI=10). After a 1 h incubation, gentamycin (100 μg/ml) was added tothe monocyte cultures containing bacteria and the incubation was thencontinued for another 2 h, making a total of a 3 hour incubation. TheRNA was then extracted and real-time RT PCR performed. In the case ofevery single stimulant used, generation 3 polypropyletherimine anioniccarboxylic acid terminated dendrimer glucosamine lead to a significantreduction in the pro-inflammatory cytokines IL-6, TNF-alpha, and IL-8.

This figure summarises the results for TNF-alpha. They show thefollowing reductions:—(1) Salmonella LPS—an 20-fold reduction at 100μg/ml; (2) live E. coli bacteria—a 6-fold reduction at 100 μg/ml; (3)live Pseudomonas aeruginosa bacteria—a 3-fold reduction at 100 μg/ml;(4) live Klebsiella pneumonia bacteria a 12-fold reduction at 200 μg/ml;(5) live S. aureus bacteria—a 10-fold reduction at 100 μg/ml; (6) liveE. faecalis bacteria—a 45-fold reduction at 200 μg/ml.

FIG. 14: The biological effect of generation 3 polypropyletherimineanionic carboxylic acid terminated dendrimer glucosamine was determinedin 24 well plates using 10⁶ human monocytes pretreated with 12.5 to 100μg/m1 endotoxin free dendrimer glucosamine for 1 h followed by challengewith:—(1) 25 ng/ml Salmonella LPS; (2) live E. coli bacteria(multiplicity of infection (MOI)=10); (3) live Pseudomonas aeruginosabacteria (MOI=10); (4) live Klebsiella pneumonia bacteria (MOI=10); (5)live S. aureus bacteria (MOI=10); (6) live E. faecalis bacteria(MOI=10). After a 1 h incubation, gentamycin (100 μg/ml) was added tothe monocyte cultures containing bacteria and the incubation was thencontinued for another 2 h, making a total of a 3 hour incubation. TheRNA was then extracted and real-time RT PCR performed. In the case ofevery single stimulant used, generation 3 polypropyletherimine anioniccarboxylic acid terminated dendrimer glucosamine lead to a significantreduction in the pro-inflammatory cytokines IL-6, TNF-alpha, and IL-8.

This figure summarises the results for MIP-1 beta. They show thefollowing reductions:—(1) Salmonella LPS—a 70-fold reduction at 100μg/ml; (2) live E. coli bacteria—a 5-fold reduction at 300 μg/ml; (3)live Pseudomonas aerugenosa bacteria—a 3-fold reduction at 100 μg/ml;(4) live Klebsiella pneumonia bacteria—a 30-fold reduction at 200 μg/ml;(5) live S. aureus bacteria—a 5-fold reduction at 100 μg/ml; (6) live E.faecalis bacteria—a 35-fold reduction at 200 μg/ml.

FIG. 15: Shows the H-NMR spectrum for the polypropyletherimine core with16 terminal carboxylic acids.

FIG. 16: Shows the 2-dimensional H-COSY spectrum for thepolypropyletherimine core with 16 terminal carboxylic acids.

FIG. 17: Shows the ¹³C-NMR spectrum for the polypropyletherimine corewith 16 terminal carboxylic acids.

FIG. 18: Shows the Distortionless Enhancement by Polarization Transfer135 ¹³CNMR spectrum for the polypropyletherimine core with 16 terminalcarboxylic acids.

FIG. 19: Shows the MALDI mass spectrum for the polypropyletherimine corewith 16 terminal carboxylic acids.

FIG. 20: Shows the HPLC charged aerosol detection trace for thepolypropyletherimine core with 16 terminal carboxylic acids.

FIG. 21: Shows a diagrammatic representation of PETIM conjugation toglucosamine

FIG. 22 a: Shows the H-NMR spectrum for the polypropyletheriminedendrimer glucosamine (with 16 terminal carboxylic acids).

FIG. 22 b: Shows the ¹³C-NMR spectrum for the polypropyletheriminedendrimer glucosamine (with 16 terminal carboxylic acids).

FIG. 23: Shows the HPLC-UV trace for the polypropyletherimine dendrimerglucosamine (with 16 terminal carboxylic acids).

FIG. 24: Show polypropyletherimine-glucosamine is not cytotoxic toprimary human monocytes.

FIG. 25: Shows high purity (95%) polypropyletherimine-glucosamine testedusing human monocytes and shigella LPS was bioactive at 50 μg/ml.

FIG. 26: Shows polypropyletherimine-glucosamine tested using humanmonocytes and infectious E. coli bacteria was bioactive at 100 μg/ml.

FIG. 27: Shows polypropyletherimine-glucosamine after storage at 37° C.& 100% humidity in a sealed vial (under argon and moisture free) for 42days was still bioactive at 100 μg/ml when tested using human monocytesand salmonella LPS.

FIG. 28: Shows Polypropyletherimine-Glucosamine does not haveAntibacterial Properties

Statement as to US Government Sponsored Research:

This invention was made, in part, with US Government funds from theNational Institutes of Health. The US Government has certain rights inthe invention.

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1. A glycodendrimer comprising: a) a non-toxic dendrimerpolypropyletherimine core supporting 16 terminal carboxylic acid groups,and b) conjungated to said core 2, 3, 4 or 5 glucosamine molecules,wherein each glucosamine is linked directly through a zero length amidebond with a residue of a terminal carboxylic acid group.
 2. Aglycodendrimer according to claim 1 comprising 2, 3 or 4 glucosaminemolecules.
 3. A glycodendrimer according to claim 1 wherein theglycodendrimer is a generational dendrimer core.
 4. A glycodendrimeraccording to claim 3, wherein the dendrimer core is a generation
 3. 5. Aglycodendrimer according to claim 1, wherein the dendrimer core has acentral atom which is oxygen.
 6. A glycodendrimer according to claim 1,wherein the dendrimer core has a central atom which is nitrogen.
 7. Apopulation of glycodendrimers wherein the average properties of thepopulation are as defined in claim
 1. 8. A pharmaceutical formulationcomprising a glycodendrimer as defined in claim 1 or a population ofglycodendrimers wherein the average properties of the population are asdefined in claim 1 and a pharmaceutically acceptable excipient.
 9. Aformulation according to claim 8, which is formulated for topicaladministration, for infusion or direct injection, or for oraladministration.
 10. (canceled)
 11. (canceled)
 12. A formulationaccording to claim 8, wherein one dose contains in range of 10 μg to 1 gof glycodendrimer as defined in claim 1 or a population ofglycodendrimers wherein the average properties of the population are asdefined in claim
 1. 13-23. (canceled)
 24. A method of treatment,comprising administering to a patient in need thereof a therapeuticallyeffective amount of a glycodendrimer according to claim 1, a populationof glycodendrimers wherein the average properties of the population areas defined in claim 1, or a formulation comprising the glycodendrimer orthe population.
 25. A method according to claim 24, wherein theglycodendrimer, population or formulation is administered for thetreatment of a disease or condition that is associated with an excessivepro-inflammatory cytokine response in the patient.
 26. A methodaccording to claim 25, wherein the response is mediated by increasedlevels of one or more cytokines selected from the group consisting ofIL-6, TNF-α, IL-8, IL-1 β, and MIP-1 α and β.
 27. A method according toclaim 25, wherein the pro-inflammatory cytokine response is associatedwith a Gram negative and/or a Gram positive bacterial infection.
 28. Amethod according to claim 27 wherein the Gram negative infection isassociated with diarrhoea.
 29. A method according to claim 27, whereinthe infection is caused by Eschericia coli, Klebsiella aeruginosa,Staphylococcus aureus, Eschericia faecalis, and/or Pseudomonasaerugenos.
 30. A method according to claim 25, wherein thepro-inflammatory cytokine response is caused by inflammatory boweldisease.
 31. A method according to claim 30, wherein the inflammatorybowel disease is associated with an excessive stimulation of Toll Likereceptors by gut bacteria.
 32. A method according to claim 25, whereinthe pro-inflammatory cytokine response is caused by an inflammatoryrespiratory response.
 33. A method according to claim 24, wherein theglycodendrimer, population or formulation is administered in thetreatment or prophylaxis of excessive scarring during wound healing,keloid formation, eczema. or psoriasis.
 34. A method according to claim33, wherein the excessive scarring happens during wound healing isfollowing a transplant.
 35. A method according to claim 25, wherein thepro-inflammatory cytokine response is due to gingivitis.